Chemokine expression in sera of patients with microscopic polyangiitis and granulomatosis with polyangiitis

We evaluated chemokine expression and its correlation with disease activity in patients with microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) (MPA/GPA). Serum CCL2, CCL4, CCL19, CXCL1, CXCL2, and CX3CL1 level in 80 patients were analysed using multiple enzyme-linked immunosorbent assays. Correlations between variables were investigated using Pearson’s correlation analysis, and receiver operator curve analysis was performed to identify optimal CX3CL1 values in determining active disease. Multivariate logistic regression analysis was done to evaluate predictors of active disease. CCL4 (r = 0.251, p = 0.025), CXCL1 (r = 0.270, p = 0.015), and CX3CL1 (r = 0.295, p = 0.008) significantly correlated with BVAS, while CX3CL1 was associated with five-factor score (r =  − 0.290, p = 0.009). Correlations were revealed between CCL2 and CCL4 (r = 0.267, p = 0.017), CCL4 and CXCL1 (r = 0.368, p < 0.001), CCL4 and CXCL2 (r = 0.436, p < 0.001), and CXCL1 and CXCL2 (r = 0.518, p < 0.001). Multivariate analysis revealed serum CX3CL1 levels > 2408.92 pg/mL could predict active disease (odds ratio, 27.401, p < 0.001). Serum chemokine levels of CCL4, CXCL1, and CX3CL1 showed association with disease activity and especially, CX3CL1 > 2408.92 pg/mL showed potential in predicting active MPA/GPA.


Statistical analyses
Continuous data are presented as medians (interquartile ranges), and categorical variables are presented as numbers (percentages).Differences in variables were evaluated using the Mann-Whitney U test and Kruskal-Wallis test for continuous variables and the chi-square test or Fisher's exact test, as indicated.The interrelationship between continuous variables was assessed using Pearson's correlation analysis, and receiver operator curve (ROC) analysis using the Youden index was used to derive the optimal CX3CL1 cut-off for predicting active disease.Multivariate logistic regression analysis using a forward entry method, including variables of statistical significance in the univariate analysis, was conducted to confirm the association between CX3CL1 and disease activity.In all statistical analyses, a two-tailed p-value of < 0.05 was considered statistically significant using MedCalc statistical software version 22 (MedCalc Software Ltd., Ostend, Belgium).

Ethical approval
This study was performed in accordance with the principles set forth in the 1964 Helsinki Declaration and its later amendments and comparable standards and was approved by the Institutional Review Board of Severance Hospital (4-2016-0901).

Characteristics of patients with MPA and GPA
Table 1 shows the clinical characteristics of the patients included in this study.Compared with patients with GPA, those diagnosed with MPA were older, had a shorter disease duration, and had higher BVAS and FFS.ENT involvement was more common in patients with GPA, whereas renal involvement was frequently observed in patients with MPA.In regard to laboratory tests, patients with MPA had lower haemoglobin and albumin levels than those in the GPA group.Concerning the expression of chemokines in patient sera, CX3CL1 expression was significantly higher in patients with MPA than in those with GPA; however, there was no difference in the expression of other chemokines according to patient diagnosis (Table 1).On the other hand, when we categorized the patients into active and inactive disease, 48 and 32 were included in the active and inactive disease group, respectively.Patients with active disease more frequently demonstrated renal manifestation compared to those without and the majority of laboratory variables investigated revealed significant differences between the groups, with an exception of AST and ALT (Table 2).
Next, when the patients were divided into active and inactive disease and active disease and remission according to the BVAS cut-off of 12 and EULAR definition, respectively, the expression of CCL2 was lower and CX3CL1 was higher in patients with BVAS ≥ 12 than those with BVAS < 12, whereas the difference was only noted for CCL2 in patients with active disease and remission based on the EULAR definition.Moreover, on categorizing patients into the disease duration of 3 months and ANCA groups, the expression of chemokine CX3CL1 was significantly higher in the disease duration < 3 months group.In contrast, CXCL1 expression was higher in patients with a disease duration of < 3 months and in those with ANCA positivity (Table 3).A comparison of CX3CL1 levels according to the pattern of organ involvement indicated that patients with renal involvement had significantly higher CX3CL1 levels than those without (p < 0.001) (Fig. 1).

Discussion
Chemokines play a crucial role in coordinating immune cell migration towards inflamed tissues, leading to their activation 39 .Consequently, chemokines produced by various cell types in response to pro-inflammatory signals serve as key mediators that bridge the innate and adaptive immune systems. 40In this study, we observed a difference in the expression of CX3CL1 and CXCL1 in sera of patients with MPA and GPA.Additionally, CCL4, CX3CL1, and CXCL1 were significantly associated with disease activity as assessed using the BVAS, and only CX3CL1 correlated with FFS.The relationship between chemokines and organ damage, estimated using Figure 1.Serum CX3CL1 levels according to organ involvement.Patients with renal involvement showed significantly higher CX3CL1 levels than those without renal involvement.There were no differences in CX3CL1 levels according to the presence or absence of ENT, nervous system, or cardiac involvement.ENT, ear, nose, and throat.the VDI, was not evident.Notably, a CX3CL1 level > 2408.92pg/mL was an independent predictive factor of active disease even when laboratory tests that are generally performed during routine patient care were taken into account, indicating that CX3CL1 could serve as a potential biological marker for the evaluation of disease activity in MPA/GPA.Several factors may indicate an association between chemokine expression and disease activity in MPA/GPA.Heightened inflammation in MPA/GPA can be reflected in various factors, including chemokine upregulation.An increase in chemokine levels can occur because of enhanced cytokine production and polarization of helper T and B cell subsets, corresponding to greater levels of inflammation.A characteristic feature of MPA/GPA is an imbalance in cytokine production, with cytokines, such as tumour necrosis factor-alpha, interleukin (IL)-6, and IL-17, implicated in disease pathogenesis 41 .These cytokines stimulate chemokine production, thereby promoting immune cell recruitment and inflammation 42 .Additionally, the expansion of Th1 and Th17 helper T cell subsets, which play a pivotal role in MPA/GPA, leads to the release of inflammatory cytokines that further induce chemokine production, thereby amplifying pro-inflammatory signalling in the disease 43 .Furthermore, B cells specifically contribute to MPA/GPA by producing ANCA antibodies 44 .Abnormal activation and differentiation of B cells can enhance disease activity by overexpressing pathogenic antibodies and cytokines such as B cell-activating factors, thereby promoting chemokine production and exacerbating inflammation 45 .Lastly, activated monocytes and macrophages at the site of inflammation also contribute to the perpetuation of inflammatory cycle and damage in MPA/GPA 46 .Conversely, increased chemokine expression in MPA/GPA intrinsically influences the recruitment and infiltration of specific leukocyte subsets into affected tissues 47 .As these immune cells contribute to inflammatory processes within blood vessels, this could result in tissue injury in MPA/GPA.
To date, various studies have reported the expression of circulating chemokines in patients with AAV.Recent publications have demonstrated that CXCL9 and CXCL16 are increased in AAV compared to controls 23,25 .In addition, a distinct chemokine levels in the circulation was also identified in PR3-ANCA AAV according to organ involvement 21 , and a prognostic role of CCL18 in ANCA-associated glomerulonephritis has been also described 22 , with the disease activity status predicting potential of CCR8, CXCL2, CXCL13, CCL5, CCL20, CCL22 suggested in AAV 24,[26][27][28][29] .On the other hand, investigation of CX3CL1 in patients with MPA and GPA revealed increased level in the periphery compared to controls, with relationship present with BVAS and active disease, respectively 30,31 .Nevertheless, due to significant discrepancies in defining AAV and disease status, differences in evaluated AAV subtypes and organ involvement, and the restricted patient sample size, the clinical implications of assessing chemokines in the peripheral blood of patients with AAV is still unclear which deserve better understanding.
This study revealed that of the six chemokines examined, only CX3CL1 levels were independently associated with disease activity in MPA/GPA.CX3CL1, also referred to as fractalkine, may serve as an indicator of ongoing inflammation in MPA/GPA because it may potentially stimulate the activation and recruitment of neutrophils, which play a crucial role in the development of MPA/GPA 48 .Elevated levels of CX3CL1 augment the attachment Table 6.Logistic regression analysis for the prediction of active disease.*CX3CL1 included in the multivariate analysis.**CX3CL1 2408.92> pg/mL included in the multivariable analysis.ANCA: anti-neutrophil cytoplasmic antibody, WBC: white blood cell, CRP: C-reactive protein, ESR: erythrocyte sedimentation rate, BUN: blood urea nitrogen, AST: aspartate aminotransferase, ALT: alanine aminotransferase.www.nature.com/scientificreports/ of neutrophils to endothelial cells, facilitating their movement into inflamed tissues and causing tissue damage.Additionally, CX3CL1 signalling through its receptor CX3CR1 can initiate intracellular signalling pathways in immune cells, leading to the generation of pro-inflammatory cytokines and chemokines and contributing to the observed inflammatory response 49 .Furthermore, CX3CL1 influences T cell migration, activation, and production of T cell-related cytokines, potentially contributing to the immune dysregulation observed in MPA/GPA 50 .Alternatively, increased levels of CX3CL1, which is expressed in endothelial cells, may indicate endothelial activation 51 .Activated endothelial cells upregulate adhesion molecules, facilitate leukocyte recruitment, and perpetuate inflammation in patients with MPA/GPA.Supporting these hypotheses, previous studies have indicated the involvement of CX3CL1 in various type of vasculopathies 30,31,52,53 ; of note, our findings showed that patients with MPA had higher levels of CX3CL1 compared to those with GPA.Even though the elevated BVAS in patients with MPA compared to those with GPA may account for this observation, given that previous research indicated the involvement of the CX3CL1-CX3CR1 axis in organ damage in MPO-ANCA-associated vasculitis 54 , it is plausible that CX3CL1 may play a greater role in promoting inflammation in MPA, where MPO-ANCA and renal involvement is more frequently observed.
The results of our study suggest that CX3CL1 could potentially be used as a biomarker to assess disease activity in MPA/GPA.Monitoring CX3CL1 levels could offer healthcare providers useful information regarding the extent of the inflammatory response and severity of the disease.It is worth noting that in addition to laboratory measures, such as WBC count and albumin levels, a CX3CL1 level of 2408.92 pg/mL was a predictive factor for active disease status.Furthermore, the correlation between CX3CL1 and FFS indicates that CX3CL1 may also have prognostic implications as FFS is reportedly related to adverse clinical outcomes in patients with AAV 35 .Therefore, incorporating CX3CL1 testing into clinical practice may be beneficial for providing optimal care to patients with MPA/GPA, with increased attention required for those exhibiting higher CX3CL1 levels.Additionally, considering that blocking CX3CL1 has shown efficacy in reducing inflammation in autoimmune disorders and potential for treating RA, it is plausible that targeting CX3CL1 could also have beneficial effects in managing MPA/GPA 55 .However, owing to the limitations of this cross-sectional study, we were unable to address whether assessing CX3CL1 could be useful in monitoring treatment response and predicting disease-related outcomes.Further investigations are necessary to explore these aspects.
The limitations of this study included the fact that both incident and prevalent cases of MPA/GPA were included in the analysis, and adjustment for the treatment regimen could not be performed.Furthermore, while the present study showed that CX3CL1 might be a biomarker of AAV, additional longitudinal assessments are required to verify its advantage in the follow-up of patients with MPA/GPA.Finally, the pathogenic mechanism linking CX3CL1 to inflammation in MPA/GPA remains to be elucidated in future studies.
In conclusion, our study demonstrated that among serum chemokines, CX3CL1 correlated with disease activity and was independently associated with active disease in patients with MPA/GPA.Hence, our findings suggest that evaluating CX3CL1 levels could have clinical implications for predicting disease severity.

Figure 2 .
Figure 2. Receiver operator curve analysis for the ideal cut-off value of CX3CL1 in predicting active disease.The derived cut-off value for CX3CL1 in determining active disease was 2408.92 pg/mL.AUC, area under the curve.

Table 3 .
Comparison of chemokines levels according to disease status, disease duration, and ANCA positivity.ANCA: anti-neutrophil cytoplasmic antibody, BVAS: Birmingham vasculitis activity score, EULAR: European League Against Rheumatism.Chemokines Disease

Table 4 .
Inter-correlation between the evaluated chemokines.Values indicate correlation coefficient and p-values.

Table 5 .
Correlation of chemokines and clinical parameters.